To grow beyond minimal size, tumors must generate new blood vessels. VEGF-A, expressed by most malignant tumors is primarily responsible for tumor angiogenesis and has the unusual property among angiogenic factors of activating the Ca2+ cascade and inducing vascular hyperpermeability. Hyperpermeability is a characteristic feature of newly formed tumor blood vessels and has an important role n tumor angiogenesis and stroma formation. The new blood vessels induced by VEGF-A-secreting tumors are of at least 6 distinct types and, using an adenoviral vector engineered to express VEGF-A164, we have been able to generate surrogate forms of each vessel type in a variety of normal mouse tissues. Of the several types of tumor blood vessels, mother vessels (MV) are the first to form and are the primary hyperpermeable vessel subset. MV are greatly enlarged, pericyte-poor sinusoids that overexpress VEGFR-1 and -2; they are also the commonest type of blood vessel found in many rapidly growing mouse tumors, and are also common in human tumors. We have found that much of VEGF-A's angiogenic activities are mediated through an orphan nuclear transcription factor, TR3 (human)/Nur77 (mouse). Our overall hypothesis is that TR3/Nur77 has a central, essential role in regulating VEGF-A-induced vascular permeability, vascular ultrastructure and the earliest phase of tumor angiogenesis that leads to the formation of hyperpermeable mother vessels. Two Specific Aims will test this central hypothesis: Aim 1. Quantify the effects of Nur77 expression levels on vascular permeability, vascular ultrastructure and angiogenesis, making use of wild type and engineered Nur77-/- and transgenic Nur77-S mice, and Aim 2. Elucidate the signaling pathways that activate Ca2+ and that regulate TR3 expression and transcriptional activity in cultured endothelial cells and in Matrigel plug assays in vivo. These Aims make use of different technologies to focus on a single objective, elucidating the steps and mechanisms by which VEGF-A induces TR3/Nur77 expression and activation, and so angiogenesis and associated vascular hyperpermeability. Relevance to public health: These studies will clarify the mechanisms by which tumors initiate the formation of the new blood vessels they require for growth and survival and, in the process, identify new potentia targets and points of intervention for attacking the tumor vasculature.